The broad goal of this Project is to utilize a unique and novel shared resource-access to fresh tissue from organ donors-to characterize B cell compartments, subsets, and responses in the normal human immune system. We will emphasize tissue-resident cells, including in lymphoid tissue, and cells resident in mucosal and parenchymal tissues. The first goal of the project is to use flow cytometric and immunohlstologic analysis to comprehensively determine the phenotypes, subsets, and characteristics of B lineage cells in all of the above tissues. In addition to the discovery of new B cell subsets-a major endpoint-this effort will provide a unique atlas of B cells in the human, and will greatly aid correlation and interpretation of data on PBL, which is often the only available sample from human subjects. The second goal is to use to define gene expression profiles of key populations identified in the first part of the work. This will substantiate the definition of phenotypic subsets, allow for meaningful comparison between analogous subsets in different tissues as well as definition of tissue-specific adaptations (e.g. chemokine receptors specific to one tissue or another among otherwise similar types of B cells). It will also provide many insights into the function of both newly discovered and previously known B cell types; gene expression and phenotype will allow alignment with better-studied analogous murine cell types, strengthening both human and murine lines of work. Again, a wealth of information useful to practically all human immune research will result. Finally, we will take advantage of unique properties of lymphocytes-clonal VDJ rearrangements-and in particular of B cells- somatic hypermutation-to obtain a dynamic picture from an otherwise static analysis. This will be enabled by the exponentially expanding capability of deep sequencing, and we will sample literally millions of V regions in a single run. VDJ-based clonal markers will allow us to trace clones between both tissues and B cell subsets. Adding information about somatic hypermutation and the clonal trees they imply will determine lineage distance between sites as well as whether local immune responses are taking place within subclones. The latter will be bolstered by selective laser capture microdissection and sequencing studies. All of these studies will be targeted to the specific subsets of cells defined by flow and gene expression in the first two phases. Further dividends from this last phase will come from analysis of mutation patterns (e.g. R/S) which will quantitate selection as well as determine whether novel cell types are Ag-experienced. We will not only deeply explore individual humoral immune systems but also define the extent of individual variation, providing initial insights into the diversity in the nature of responses to vaccines and pathogens.